Purification and properties of multiple forms of mouse trypsinogen

Three trypsinogens and one chymotrypsinogen were found in and purified from the pancreas of a mouse strain (CFO). The molecular weights of the trypsinogens and the chymotrypsinogen were all estimated as 25 000. The enzyme properties of the three trypsinogens were studied and they showed very low K_m values (3.2?6.5 μM) for the substrates, BzArgOEt and TosArgOMe, and the dame pH optimum profile between pH 8.0–10.0. However, the ratios of catalytic rate constants, k_cat (s^?1), with BzArgOEt as substrates compared to that with TosArgOMe were very different. The values of Try-III were similar with the two substrates, Try-I was slightly higher value with TosArgOMe than with BzArgOEt, and the values of Try-II were much higher with TosArgOMe than with BzArgOEt. Also, the trypsinogens and the chymotrypsinogen were purified from pancreas of Mol-A strain mice. When the enzyme properties of the three trypsinogens were examined, one form of trypsinogen (Try-I) was shown to have different properties in k_cat (s^?1) for the two substrates, compared to the trypsinogen of CFO mice.     

Catalytic role of the calcium ion in GH97 inverting glycoside hydrolase

The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme, BtGH97a, was investigated through structural and kinetic studies. The calcium ion was likely directly involved in the catalytic reaction. The pH dependence of k_cat/K_m values in the presence or absence of calcium ion indicated that the calcium ion lowered the pK_a of the base catalyst. The significant decreases in k_cat/K_m for hydrolysis of substrates with basic leaving groups in the absence of calcium ion confirmed that the calcium ion facilitated the leaving group departure.     

Key aromatic residues at subsites + 2 and + 3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k_cat/K_m for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k_cat/K_m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k_cat/K_m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k_cat/K_m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.     

Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k _cat, K _m, and k _cat/K _m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate—the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.     

Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre-Steady-State Study

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k_cat and k_cat/K_m reflect the ionization of two groups with pK_a values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K_m are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10⁻¹⁰ molar at the optimum pH value (approximately 7.5).     

Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.

Alcohol dehydrogenase SS was prepared from horse liver by salt fractionation, ion-exchange chromatography, and affinity chromatography. The purified isoenzyme is free from extraneous protein and other alcohol dehydrogenase isoenzyme contaminants and contains four Zinc atoms per molecule. The substrate specificity with saturated aliphatic alcohols and aldehydes of two to six carbon chain lengths has been investigated. The K_m values and turnover numbers at maximal velocity (k_cat) are presented. Values of k_cat are constant within a substrate category and independent of the substrate chain length, while the K_m values decrease with the increase of the substrate chain length. The K_m values for two- and three-carbon substrates are large, that for ethanol (40 mm) is two orders of magnitude larger than that reported for classical preparations of horse liver alcohol dehydrogenase. At pH 7, the k_cat values for alcohol oxidation are almost 30 times smaller than for aldehyde reduction. The enzyme has been characterized with regard to specific activity with several nonsteroidal substrates and with two steroids: 3-oxo-5β-androstan-17β-ol and 5β-pregnan-21-ol-3,20-dione hemisuccinate. NAD(H) is the preferred coenzyme. Values of K_m for NADH with constant steroidal substrates are an order of magnitude smaller than the corresponding K_m values with nonsteroidal substrates. A possible explanation for this observation is presented.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: Changes in kcat and thermal dependence

Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its k_cat and a remarkable increase in its stability. While the increase in k_cat is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, k_cat, K_M and k_cat/K_M were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis.     

Substituents effects on activity of kynureninase from Homo sapiens and Pseudomonas fluorescens

A series of substituted kynurenines (3-bromo-dl, 3-chloro-dl, 3-fluoro-dl, 3-methyl-dl, 5-bromo-l, 5-chloro-l, 3,5-dibromo-l and 5-bromo-3-chloro-dl) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have k_cat and k_cat/K_m values higher than l-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have k_cat and k_cat/K_m values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have k_cat and k_cat/K_m values comparable to that of its physiological substrate, l-kynurenine. Thus, 5-bromo and 5-chloro-l-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase.     

Identification of Residues Surrounding the Active Site of Type A Botulinum Neurotoxin Important for Substrate Recognition and Catalytic Activity

Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain (LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar residues resulted in drastic lowering of the catalytic rate constant (k _cat), but had less effect on substrate affinity (K _m). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k _cat/K _m) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation to Asn resulted in 140-fold decrease in k _cat/K _m. Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163, Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165 with Leu resulted in fourfold increase in k _cat/K _m. For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe, respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC competent for catalysis or provide conformational selection of the substrate.     

Acid carboxypeptidase from a wood-deteriorating basidiomycete, Pycnoporus Sanguineus

An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a K_m of 0.74 mM and a k_cat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The K_m and k_cat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively.     

Formulation of a Universal First-Order Rate Constant for Enzymatic Reactions

It is a common practice to employ k _cat[E]₀/K _m as a first-order rate constant for the analysis of an enzymatic reaction, where [E]₀ is the total enzyme concentration. I describe in this report a serious shortcoming in analyzing enzymatic reactions when k _cat[E]₀/K _m is employed and show that k _cat[E]₀/K _m can only be applied under very limited conditions. I consequently propose the use of a more universal first-order rate constant, k _cat[ES]_K/[S]₀, where [ES]_K is the initial equilibrium concentration of the ES-complex derived from [E]₀, [S]₀ and K _m. Employing k _cat[ES]_K/[S]₀ as the first-order rate constant enables all enzymatic reactions to be reasonably simulated under a wide range of conditions, and the catalytic and binding contributions to the rate constant of any enzyme can be determined under any and all conditions.     

Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis

The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k_cat/K_m) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in k_cat/K_m with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.     

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9₂₅-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9₂₅-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5′-product terminating in a 2′,3′-phosphodiester and a 3′-product terminating in a 5′-OH. Under single turnover conditions at 24°C, 9₂₅-11t displays a maximum first-order rate constant, k_cat, of 0.037 min⁻¹ and a catalytic efficiency, k_cat/K_m, of 5.3 × 10⁵ M⁻¹ min⁻¹. The measured value of k_cat under catalyst excess conditions agrees with the value of k_cat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9₂₅-11t was gauged in terms of k_cat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k_cat values by a factor of 4 to 250, indicating that 9₂₅-11t displays significant sequence specificity despite the lack of an apparent Watson–Crick base-pairing scheme for recognition.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Contributions of binding and catalytic rate constants to evolutionary modifications inKm of NADH for muscle-type (M4) lactate dehydrogenases

Summary The apparent Michaelis constant (K _m) of NADH for muscle-type (M₄ isozyme) lactate dehydrogenases (LDHs) is highest, at any given temperature of measurement, for LDHs of cold-adapted vertebrates (Table 1). However, these interspecific differences in theK _m of NADH are not due to variations in LDH-NADH binding affinity. Rather, theK _m differences result entirely from interspecific variation in the substrate turnover constant (k _cat) (Fig. 1; Table 2). This follows from the fact that theK _m of NADH is equal tok _cat divided by the on constant for NADH binding to LDH,k ₁, so that interspecific differences ink _cat, combined with identical values fork ₁ among different LDH reactions, make the magnitude of theK _m of NADH a function of substrate turnover number. The temperature dependence of theK _m of NADH for a single LDH homologue is the net result of temperature dependence of bothk _cat andk ₁ (Figs. 3 and 4). Temperature independentK _m values can result from simultaneous, and algebraically offsetting, increases ink _cat andk ₁ with rising temperature. Salt-induced changes in theK _m of NADH also may be due to simultaneous perturbation of bothk _cat andk ₁ (Table 3). These findings are discussed from the standpoint of the evolution of LDH kinetic properties, particularly the interspecific conservation of catalytic and regulatory functions, in differently-adapted species.     

An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches

The action of pancreatic α-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine α-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E₀] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of k_cat/K_m performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E₀ and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100°C, k_cat/K_m values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and k_cat/K_m is not a simple one. The value of k_cat/K_m rises to reach a maximum at a pre-treatment temperature of 75°C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although α-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.     

STUDIES ON THE KINETICS OF ACETYLCHOLINESTERASE IN THE RESISIANT AND SUSCEPTIBLE STRAINS OF HOUSEFLY (MUSCA DOMESTICA)

Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The V_max values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The V_max ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The K_m values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The K_m ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The k_i ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, k_cat and k_cat/K_m for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The k_cat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of k_cat/K_m were in contrary to the k_cat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of V_max, K_m, k_i, k_cat and k_cat/K_m. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides.     

Structural Advantage of Sugar Beet α-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate

The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower K_m and higher k_cat/K_m for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log K_i of the acarviosyl-maltooligosaccharides and log(K_m/k_cat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the K_m for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.